Bone protein extraction without demineralization

Abstract

Embodiments of the invention relate generally to protein extraction and, more generally, to bone protein extraction methods that do not require demineralization. In one embodiment, the invention provides a method comprising: mixing a bone sample and a quantity of an extraction buffer comprising: ammonium phosphate dibasic; or ammonium phosphate dibasic and ammonium bicarbonate; or ammonium phosphate dibasic, ammonium bicarbonate, and guanidine HCl; or sodium phosphate dibasic and sodium bicarbonate; or sodium phosphate dibasic, sodium bicarbonate, and guanidine HCl; or potassium phosphate dibasic and potassium bicarbonate; or potassium phosphate dibasic, potassium bicarbonate, and guanidine HCl; and incubating the bone sample/extraction buffer mixture.

The invention claimed is: 
     
       1. An extraction method comprising:
 mixing a bone sample and a quantity of an extraction buffer comprising:
 ammonium phosphate dibasic, ammonium bicarbonate, and guanidine HCl; 
 
 incubating the bone sample/extraction buffer mixture; 
 extracting at least one protein from the bone sample via the extraction buffer; and 
 separating the at least one protein from the bone sample/extraction buffer mixture, 
 wherein the method does not include a demineralization step incorporating a demineralization agent. 
 
     
     
       2. The method of  claim 1 , wherein the extraction buffer includes ammonium phosphate dibasic at a concentration between about 400 mM and about 1 M. 
     
     
       3. The method of  claim 1 , wherein the extraction buffer includes ammonium bicarbonate at a concentration of about 200 mM. 
     
     
       4. The method of  claim 1 , wherein the extraction buffer includes guanidine HC1 at a concentration of about 4 M. 
     
     
       5. The method of  claim 1 , wherein the extraction buffer comprises ammonium phosphate dibasic, ammonium bicarbonate, and guanidine HC1 and the at least one protein is selected from a group consisting of: alpha-1-antitrypsin, alpha-2-macroglobulin, basement membrane-specific heparan sulfate proteoglycan core protein, biglycan, chondroadherin, collagen alpha-1(I) chain, collagen alpha-1(III) chain, collagen alpha-1(XI) chain, collagen alpha-2(I) chain, collagen alpha-2(VI) chain, collagen alpha-3(VI) chain, collagen alpha-5(IV) chain, histone H2A 1-B/E, Ig gamma-1 chain C region, Ig kappa chain C region, keratin-associated protein 1-1, lumican, osteocalcin, osteomodulin, pigment epithelium-derived factor, serum albumin, tenascin, and vitronectin. 
     
     
       6. The method of  claim 1 , wherein the extraction buffer further comprises at least one protease inhibitor selected from a group consisting of: sodium fluoride, sodium orthovanadate, sodium pyrophosphate, beta-glycerophosphate, and mixtures thereof. 
     
     
       7. The method of  claim 1 , wherein separating the at least one protein includes desalting the bone sample/extraction buffer mixture with water. 
     
     
       8. The method of  claim 1 , wherein incubating includes holding the bone sample/extraction buffer mixture at at least room temperature. 
     
     
       9. The method of  claim 1 , wherein incubating includes holding the bone sample/extraction buffer mixture at a temperature between room temperature and about 75 ° C. for at least four hours. 
     
     
       10. The method of  claim 1 , wherein the extraction buffer further comprises phenacylthiazolium bromide (PTB) and the at least one protein is cathepsin K. 
     
     
       11. The method of  claim 1 , wherein the bone sample is a mineralized bone sample.